An 'All-in-One' Platform for Multiple Trait Stacking in Crops Developed by Caixia Gao's Team

A major goal of modern crop breeding is to efficiently combine multiple desirable traits — a process called "stacking" — into a single crop variety by selecting for favorable gene variants. However, current strategies are often time-consuming and inefficient.

Led by Prof. Caixia Gao at the Institute of Genetics and Developmental Biology (IGDB), Chinese Academy of Sciences, our team has developed TRIM — a unified genome engineering platform that combines gene knockout, precise sequence editing, and large-scale chromosome engineering in a single framework. The work was published in Nature Biotechnology on June 5, 2026.

As a foundation of the platform, we developed a precise and efficient gene knockout tool called twin prime editing (twinPE)-mediated gene knockout (TKO), which precisely inserts a small fragment containing a stop codon cluster at the target site. TKO achieves predictable gene disruption through precise installation of stop codons, avoiding in-frame indels caused by multiple-3n base insertions or deletions in the Cas9 systems.

In protoplasts, TKO demonstrated efficient knockout capabilities in monocot crops such as rice, wheat, and maize. In regenerated T0 rice plants, the average efficiency for single gene knockout reached 96.8%.

To support simultaneous editing and knockout at multiple loci without mutual interference, we further developed 10 orthogonal TKO systems, allowing 10 genes to be knocked out in a single operation. Orthogonal TKO systems maintain high knockout efficiency when simultaneously editing multiple genes or homologs, avoiding the loss of effectiveness seen in Cas9-mediated multiple editing caused by accumulated in-frame mutations across targets.

Building on TKO, we then developed two integrated genome engineering platforms, TRIM1 and TRIM2—forming a unified platform known as TRIM.

TRIM1 combines TKO with prime editing-based sequence modification, enabling simultaneous gene knockout, base substitution, insertion, deletion, duplication, and inversion within a single editing framework. In regenerated T0 rice plants, TRIM1 achieved simultaneous knockout of one gene together with homozygous precise editing of three additional targets with an efficiency of 22.8%.

TRIM2 incorporates a prime editor–Cre recombinase fusion protein and enables kilobase-scale DNA insertion, replacement, deletion, inversion, and chromosomal translocation through recombinase-assisted genome engineering.

Whereas conventional genome editing tools are typically restricted in the number of sequence modifications they can execute, TRIM consolidates gene knockout, localized precise editing, and large-scale chromosome engineering into a single platform. This comprehensive capability provides a powerful method for rapidly stacking multiple favorable alleles to enhance precision breeding in monocot crops.

Figure 1. Development of the precise gene knockout TKO system and the all-in-one TRIM platforms. a–c, Schematic of TKO (a), TRIM1 (b), and TRIM2 (c). d–f, Editing efficiencies of TKO (d), TRIM1 (e) and TRIM2 (f) at representative target sites.